Method of preparation of compositions comprising dehydrated micro-organisms

ABSTRACT

The invention relates to a composition comprising revivable dehydrated micro-organisms. The invention is characterized in that it further comprises particles at least 50% of which have a mean diameter greater than 250 μm. The invention is applicable, in particular, to human or veterinary pharmaceuticals, to dietetics or to food products.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 11/815,703filed Feb. 13, 2008, which is a U.S. national stage of Internationalapplication No. PCT/FR2006/000258 filed Feb. 6, 2006, each of whichclaims priority of French application No. 05 01232 filed Feb. 7, 2005,the entire contents of each of which being hereby incorporated byreference.

FIELD OF THE INVENTION

This invention relates to compositions comprising revivable dehydratedmicro-organisms, methods of preparing such compositions, and their usesas food intake, in particular dietetic, or as a drug.

BACKGROUND OF THE INVENTION

The intestinal flora of man and animals, in particular warm-bloodedanimals, contains numerous species of micro-organisms. In particular,these are bacteria the presence of which is essential, not only todigestion, but also, and more generally speaking, to health.

Therefore, numerous food products or drugs have been developed with aview to providing micro-organisms capable of reconstituting theintestinal flora of man and animals.

In manufacturing such food products or such drugs, the micro-organismsare generally dehydrated in advance. They are further associated withcompounds, said compounds advantageously having prebiotic properties,i.e., being capable of stimulating the growth of micro-organisms.

However, the dehydrated micro-organisms of these food products aresubjected to stresses. Furthermore, these are stresses which are notnecessarily due to the environment. Chemical stresses such as acidity,or enzymatic stresses may be cited, for example.

The effect of these stresses is to affect the viability of thedehydrated micro-organisms with the result being that, for all practicalpurposes, it is not possible to guarantee the presence of a substantialquantity of revivable micro-organisms in these food products, and thisis so even when the storage life of said products is short.

Of course, it has been sought to develop compositions containingrevivable dehydrated micro-organisms in which said micro-organisms areprotected from the physicochemical stresses to which they are normallysubjected.

These compositions aim to protect the micro-organisms by coating themwith a protective substance. They are disclosed, in particular, in thedocuments published under the numbers FR 2 806 417, FR 2 748 752 and US2004/0175389. In the document FR 2 806 417, micro-organisms are coatedwith a protective hydrophobic substance. In addition, in the patent FR 2748 452, bacteria are coated with polymers. Finally, in the document US2004/0175389, bacteria are coated with an alginic acid gel.

Nevertheless, the coating of dehydrated bacteria is an operation whichis stressful in itself, and which consequently ends up destroying asubstantial quantity of the bacteria present in the compositionsobtained. Furthermore, coating is a relatively complex and randomoperation from product to product.

SUMMARY OF THE INVENTION

In light of the foregoing, one problem the invention proposes to resolveis to produce a composition comprising revivable dehydratedmicro-organisms, as well as a method for preparing such a composition,which introduces a certain stability of the micro-organisms over time.

The first object of the solution of the invention to this posed problemis a composition containing revivable dehydrated micro-organisms,characterised in that it further comprises particles 50% of which have amean diameter greater than 250 μm.

The second object is a method for preparing a food or medicinalcomposition, characterized in that it comprises the following steps of:

-   -   dehydrating revivable micro-organisms; and    -   mixing the dehydrated micro-organisms with particles at least        50% of which have a mean diameter greater than 250 μm.

Finally, the third object is the use of a composition such as the oneabove, as a food intake or drug for human or veterinary use.

Surprisingly, when a composition includes both dehydratedmicro-organisms and particles at least 50% of which have a mean diametergreater than 250 μm, a substantial quantity of dehydratedmicro-organisms, in actual practice more than 20%, remains revivable fora minimum of 18 months.

MODE(S) FOR CARRYING OUT THE INVENTION

The invention will be better understood upon reading the followingnon-limiting description.

The composition according to the invention comprises micro-organisms andparticles.

The micro-organisms of the composition according to the invention aremicroscopic-sized eukaryotic or prokaryotic unicellular organisms. Theseare preferably bacteria or yeasts, and more preferably bacteria oryeasts having probiotic properties i.e., useful to the organism of thehuman body or to the animal organism.

Among the bacteria capable of entering into the compositions accordingto the invention, bacteria of the family Lactobacillaceae,Bifidobacteriaceae, Streptococcaceae, Enterococcaceae or Bacillaceaewill advantageously be chosen. The following species can be cited asnon-limiting examples of bacteria of the family Lactobacillaceae:Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillusbrevis, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacilluscasei rhamnosus, Lactobacillus delbrueckii, Lactobacillus farciminis,Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillushelveticus, Lactobacillus johnsonii, Lactobacillus lactis, Lactobacillusparacasei, Lactobacillus plantarum, Lactobacillus pentosaceus,Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivariusand Pediococcus acidilactici. The following species can be cited asnon-limiting examples of bacteria of the family Bifidobacteriaceae:Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacteriumbreve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacteriumlongum and Bifidobacterium pseudolongum.

The following species can be cited as non-limiting examples of bacteriaof the family Streptococcaceae: Streptococcus infantarius, Streptococcusthermophilus, Streptococcus salivarius, and Lactococcus lactis. Thespecies Enterococcus faecium can be cited as a non-limiting example ofbacteria of the family Enterococcaceae. The following species can becited as non-limiting examples of bacteria of the family Bacillaceae:Bacillus cereus, Bacillus licheniformis, Bacillus subtilis.

Among the yeasts capable of entering into the compositions of theinvention, yeasts of the family Saccharomycetaceae will advantageouslybe chosen. The genera Saccharomyces, such as Saccharomyces boulardii andSaccharomyces cerevisiae and Kluyveromyces can be cited.

The micro-organisms according to the invention are dehydrated. The sizeof said micro-organisms, after dehydration, is generally less than 50μm. They are obtained via conventional methods such as grinding afterlyophilization, and via spray drying or air-fluidized bed drying ofrelatively concentrated and relatively pure micro-organism biomasses.

Furthermore, the micro-organisms according to the invention arerevivable. Correspondingly, they have the capacity to resume theactivity of life after a period of anhydrobiosis.

The particles of the composition according to the invention are capableof including any constituent, alone or in a mixture, which is used inmanufacturing or preparing a food or medicinal composition. Inparticular, these are vitamins, trace elements, minerals and the saltsthereof, carriers such as sugars or amino acids or vegetable flours,alone or in a mixture, introducing various properties into saidcomposition, e.g., physical properties, such as its solubilisation in anaqueous medium or effervescence in the presence of such a medium,therapeutic properties, assistance in re-growth, a nutritionalsupplement or palatability. These can also be pharmaceutical activeprinciples, e.g., such as antibiotics, anti-inflammatories,anti-parasitic agents, antacids, analgesics or tranquilizers. Theparticles are generally substantially devoid of micro-organisms.

Non-limiting examples of vitamins that can be cited include vitamin A,vitamin B1, vitamin B2, vitamin B3, vitamin B4, vitamin B5, vitamin B6,vitamin B8, vitamin B9, vitamin B12, vitamin BT, vitamin Bx, vitamin C,vitamin D, vitamin D2, vitamin D3, vitamin E, vitamin F, vitamin G,vitamin H, vitamin K, vitamin K3, vitamin M, vitamin P, vitamin P4 andvitamin PP. Non-limiting examples of trace elements that can be citedinclude calcium, cobalt, copper, iron, fluorine, iodine, magnesium,manganese, selenium and zinc. A non-limiting example of a source ofminerals that can be cited includes, in particular, calcium chloride,potassium chloride, copper gluconate, sodium fluoride, potassium iodide,manganese sulphate, magnesium stearate, zinc sulphate and ferroussuccinate. A non-limiting example of a carrier that can be citedincludes carrier sugars and agents such as fructans, in particularinulin, fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS),or trans-galacto-oligosaccharides (TOS), xylooligosaccharides (XOS),certain maltodextrins, polydextroses, lactulose, amino acids and foodfibbers. The vegetable flours, for example, are residues of oleaginousseeds or fruits, from which the oil was extracted.

In addition, the particles of the composition according to the inventionadvantageously contain effervescent agents consisting of a mixture of atleast one acid and one alkaline agent. Non-limiting examples of acidsthat can be cited include tartaric acid, citric acid, maleic acid,fumaric acid, malic acid, adipic acid, succinic acid, lactic acid,glycolic acid, alpha-hydroxy acids, ascorbic acid and the amino acids,as well as the salts and derivatives of these acids. Non-limitingexamples of alkaline agents that can be cited include potassiumbicarbonate, sodium bicarbonate, potassium carbonate, sodium carbonate,calcium carbonate, ammonium carbonate, L-lysine carbonate, argininecarbonate, glycine sodium carbonate or sodium amino acid carbonates.

The aforesaid constituents are generally in a solid form. The particlesare dried, at least at their surface, and are quite often spherical,ovoid or elongated in shape. Their surface is evenly or unevenly smoothor rough.

According to the invention, at least 50% of the particles have a meandiameter greater than 250 μm and less than 2,000 μm, preferably 1,000μm. They are distributed within a particle-size range almost exclusivelybetween 20 and 2,000 μm, and preferably between 50 and 1,000 μm.

The particles can be obtained via granulation and, consequently, are inthe form of granules. Granulation such as this can be carried out viamixing in a wet medium, and includes drying and then sizing steps. Itcan also be carried out in a fluidized-air bed or on devices known inthe prior art, which combine a set of operations resulting in thedesired particles, via compacting, via granulation or via coating. Inthis final alternative, the particles are coated.

The composition according to the invention can further contain foodadditives such as preservatives, anti-oxidants, supports, acidifiers,acidity regulators, anti-caking agents, anti-foaming agents,emulsifiers, flavour enhancers, foaming agents, gel-forming substances,coating agents, humectants, modified starches, sequestering agents,stabilisers, thickeners and water retention agents.

The composition according to the invention, for example, is the resultof a simple mechanical mixing of dehydrated micro-organisms andparticles as described above. In actual practice, it preferablycomprises between 1 and 80% w/w (weight of the total weight of thecomposition) dehydrated micro-organisms, and between 20 and 99% w/wparticles and, more preferably, between 10 and 40% w/w dehydratedmicro-organisms and between 60 and 90% w/w particles.

In addition, the composition according to the invention is dry, i.e.,unsaturated or slightly saturated with a liquid and, in particular, withwater. It preferably has a water activity lower than 0.3 and, morepreferably, a water activity lower than 0.2.

The composition according to the invention is in any pharmaceutical formnormally used for oral administration. Thus it can be in powder form andplaced in pouches, jars or bags, said composition possibly beingeffervescent, and intended to be dissolved in water. It can also be inthe form of capsules or gelatine capsules, possibly enteric, with a hardor soft casing, or else, for example, in the form of tablets orprolonged-release, coated or uncoated, buccal, sublingual oreffervescent granules, for example.

In actual practice, the compositions according to the invention can bekept moisture-free and at room temperature for a relatively long timeperiod. Surprisingly, the quantity of revivable micro-organisms in thesecompositions at the end of this time period is well above the quantityof micro-organisms that it would have been possible to obtain incompositions of the prior art, kept under the same conditions. Thus, ifit is considered that at a time=0, corresponding to its packaging, acomposition according to the invention contains 100% revivablemicro-organisms, then, after 18 months, a composition such as this, ifkept moisture-free and at room temperature, will contain at leastapproximately 20% revivable micro-organisms, after counting the numberof micro-organisms (by counting the colonies obtained after inoculationinto or onto an appropriate culture medium). This appears to besignificant with regard to the compositions of the prior art.

In a preferred embodiment according to the invention, the composition issoluble or dispersible in an aqueous medium. It can also beeffervescent. Therefore, when it is added to water, e.g., intended forfeeding large animals such as horses or cattle, it dissolves ordisperses. Effervescence facilitates the dissolution or dispersion ofthe composition in water and its diffusion within this aqueous medium.The revivable micro-organisms present in the composition are rehydrated.Rehydration is a fast step lasting a few minutes. At the end of thesefew minutes, large animals, absorbing the water in which the compositionis dissolved or dispersed, ingest rehydrated micro-organisms as well asingredients useful to their body, such as sugars, vitamins orprebiotics, if such elements are present in the original composition.

However, it is well understood that the composition according to theinvention is capable of being used for purposes other than for feedinglarge animals. As a matter of fact, it is capable of serving to feedhumans and any animal, in particular warm-blooded animals. Itconstitutes a food product or drug for human or veterinary use. Thechoice of a micro-organism or of a mixture of micro-organisms isdetermined by the end use of the composition according to the invention.

The method for preparing a food or medicinal composition according tothe invention is characterised in that it comprises the steps of:

-   -   dehydration of revivable micro-organisms; and    -   mixing the dehydrated micro-organisms with particles at least        50% of which have a mean diameter greater than 250 μm.

Dehydration is carried out according to the conventional methodsdescribed above, and the mixing with particles at least 50% of whichhave a mean diameter greater than 250 μm is carried out according tocommon known techniques.

The method of preparation is preferably carried out under controlledmoisture conditions ensuring a water activity lower than 0.3% in thecomposition obtained.

This invention will now be illustrated by means of the followingexamples.

Example 1

Particles containing vitamins A, D3, E, K, B1, B2, B3, B5, B6, B12 andC, folic acid, biotin, choline, lysine, methionine, tryptophan, citricacid, sodium carbonate and sodium bicarbonate, an absorbent and anemulsifier are prepared conventionally via rotogranulation. The particlesize is adjusted so that said particles have a particle-sizedistribution of between 50 and 1,000 μm, 65% of said particles having asize greater than 250 μm. Next, 67% by weight of the total weight (w/w)of such particles are mechanically mixed with 33% w/w of a mixture offerments containing the following micro-organisms: Lactobacillusplantarum and Streptococcus infantarius, titrating 2.52×10⁹ CFU,marketed by the Lallemand™ Company under the trade name Adjulact™. Thewater activity of the effervescent composition A thus obtained accordingto the invention is 0.17.

Example 2

Particles, which contain Enerlyte™ (marketed by the Virbac™ Company) andcitric acid, sodium carbonate and sodium bicarbonate, in order to createeffervescence, are prepared conventionally via rotogranulation. Theparticle size is adjusted so that they have a particle-size distributionof between 50 and 1,000 μm, 54% of said particles having a size greaterthan 250 μm. Next, 67% w/w of such particles are mechanically mixed with33% w/w of a mixture of ferments containing the following bacteria:Lactobacillus plantarum, Streptococcus infantarius, said mixture beingtitrated with 2.52×10⁹ CFU (Adjulact™ by Lallemand™). An effervescentcomposition according to the invention is then obtained for which themixture's water activity is 0.20.

Example 3

A tablet according to the invention is prepared, which contains 3×10⁹CFU of lyophilised Saccharomyces cerevisiae, having the formula:

Constituents Unit formula Centesimal formula Lyophilised 499.94 mg 35.71Saccharomyces cerevisiae* Mannitol 689.22 mg 49.23 Crospovidone 28.00 mg2 Povidone 56.00 mg 4 Microcrystalline 126.00 mg 9 cellulose Magnesiumstearate 0.84 0.06 TOTAL 1400.00 mg 100 Wetting liquid: Eliminated bydrying after granulation Water QS Ethanol QS *Lyophilised Saccharomycescerevisiae titrating 6 × 10⁹ CFU/g

The mannitol, crospovidone, and microcrystalline cellulose aregranulated with the hydroalcoholic solution of povidone. The granulesthus obtained are dried and sized in order to obtain a granule sizesmaller than 800 micrometers. The size of 82% of the particles is largerthan 250 micrometers.

The micro-organisms are homogenised in a ribbon blender together withthe sized granules obtained.

Tableting is then carried out with a tablet-making machine equipped withdies and punches having a diameter of 17 mm; the walls of the dies andpunches are pre-powdered with magnesium stearate, as a lubricant (thenon-adhering excess quantity of magnesium stearate is suctioned up priorto tableting).

The compressive force is of the order of 10 to 16 kN, which makes itpossible to obtain tablets preserving the structural integrity of themajority of the particles, which is estimated at more than 50%.

The amount of magnesium stearate distributed over the surface of atablet is 0.84 mg or 0.6 for one thousand.

Example 4

A comparative study of the viability of the dehydrated bacteriaLactobacillus plantarum and Streptococcus infantarius present in variouscompositions was conducted under the following conditions.

The composition A according to the invention, described in Example 1,was apportioned per 25 g into welded aluminised pouches, which offer avery good gas and moisture barrier.

In addition, a composition B was prepared, which contains exactly thesame bacteria and constituents of the particles as composition A, in thesame quantities and proportions, but a large majority of the particlesof which have a mean diameter smaller than 150 μm. Just as forcomposition A, this composition B, which is in the form of a powder, wasapportioned per 25 g into welded aluminised pouches identical to thoseof composition A.

Finally, a composition C was prepared, which contains exactly the samebacteria and constituents of the particles as compositions A and B,according to the same method as that implemented in Example 1. However,in this case, the micro-organisms are constituent parts of theparticles. The mean diameter of the particles of this composition C islarger than 300 μm. As in the preceding, this composition C wasapportioned per 25 g into welded aluminised pouches identical to thoseof compositions A and B.

Then the various pouches of the compositions A, B and C were placed inan oven kept at a temperature of 40° C. and in the presence of 75%humidity. A pouch of each of the compositions was analysed each weekwith a view to determining the revivable bacteria content of each pouch.The results are reported in Table 1 below. They are expressed in CFU/gand in percentage of the initial concentration of revivablemicro-organisms.

TABLE 1 Time 0 7 days 14 days 21 days 28 days 42 days 50 daysComposition A 4.18 × 10⁸ 3.71 × 10⁸ 3.05 × 10⁸ 2.12 × 10⁸ 1.10 × 10⁸1.10 × 10⁸ 1.10 × 10⁸ (100%) (88.75%) (72.85%) (52.72%) (26.20%)(26.20%)    (26.20%)    Composition B 2.52 × 10⁸ 1.11 × 10⁸ 1.32 × 10⁸3.54 × 10⁷ 0.21 × 10⁷ — — (100%) (43.94%) (52.18%) (14.04%)  (0.8%) (0%)(0%) Composition C 2.94 × 10⁸ 0.15 × 10⁸ 0.32 × 10⁷ — — — — (100%)   (5%)    (1%)    (0%)    (0%) (0%) (0%)

It appears that the initial concentration of viable bacteria incomposition A is higher than the initial concentration of viablebacteria in composition C, which is itself higher than the initialconcentration of viable bacteria in composition B. Accordingly, it seemsthat the steps for preparing compositions B and C produced moresignificant stresses on the bacteria than the preparation steps forcomposition A.

For all compositions, the concentration of revivable bacteria decreaseswith relation to time. However, this concentration decreases more slowlyin the case of composition A according to the invention than in theother compositions.

Furthermore, in the case of composition A, the concentration ofrevivable bacteria decreases until reaching a limit corresponding toapproximately 26.20% of the 100% of revivable bacteria initially presentin the pouches. As a result, the concentration of revivable bacteriaappears to be stabilised over time, at least up to 50 days after beingplaced in pouches under the aforesaid operational conditions.

Of course, this stability observed under stressful conditions ought tobe observed all the more so under actual conditions of storing thepouches at room temperature. Considering the fact that a week of storingthe pouches under the operational conditions of this example is equal toapproximately 3 months of storage at 25° C., it is permissible toconclude that a composition according to the invention, such ascomposition A, kept in pouches at temperatures of the order of 25° C.,ought to contain a substantial quantity of revivable bacteria, in actualpractice around 26.20% of the initial concentration, 18 months afterbeing placed in pouches. At a minimum, this example demonstrates asharply improved viability of the dehydrated micro-organisms incomparison with a composition that is substantially devoid of particleshaving a mean diameter and well below 250 μm (composition B), and incomparison with a composition in which the micro-organisms areconstituent parts of the particles (composition C).

The invention claimed is:
 1. A method of preparing a food or medicinalcomposition, wherein the method comprises: dehydrating uncoated,revivable micro-organisms, wherein the micro-organisms are a mixture ofLactobacillus plantarum and Streptococcus infantarius; and mixing thedehydrated uncoated, revivable micro-organisms with particles, wherein(i) each of said particles has a mean diameter, (ii) the mean diametersof said particles are distributed almost exclusively in a range of from50 to 1,000 μm, (iii) about 65% of said particles have a mean diametergreater than 250 μm, and (iv) about 35% of said particles have a meandiameter of at most 250 μm, so as to form a composition comprising about33% w/w revivable dehydrated micro-organisms, and further comprisingabout 67% w/w particles, wherein the micro-organisms are mixed with theparticles in the composition, wherein the particles are granulescomprising an aggregated and granulated mixture of (i) vitamins, traceelements, amino acids, sugars, and minerals or the salts thereof, and(ii) at least one carrier, and wherein the granules are substantiallydevoid of micro-organisms, wherein the composition has a water activitylower than 0.3, and wherein the micro-organisms in the composition havea revivability such that, after being kept moisture-free and at roomtemperature for 18 months, the composition comprises at least 20%revivable micro-organisms from amongst 100% revivable micro-organismsinitially present in said composition.
 2. The method of claim 1, beingcarried out under controlled moisture conditions ensuring a wateractivity lower than 0.2 in the composition obtained.
 3. The method ofclaim 1, wherein the particles contain effervescent agents, wherein theeffervescent agents comprise a mixture of at least one acid and onealkaline agent.
 4. The method of claim 1, wherein the composition issoluble or dispersible in water.
 5. The method of claim 1, wherein saidcomposition in the form of capsules or tablets.